1. Field of the Invention
The present invention relates to apparatus and method for determining diagnostically significant red blood cell parameters, i.e., hemoglobin concentration (HC) and volume (V), in a whole blood sample on a cell-by-cell basis by automated techniques. While finding particular application in the measurement of the HC and V of individual red blood cells, it will be appreciated that the present invention finds broad application in the measurement of the volume, or equivalently the diameter in the case of spherical particles, and index refraction index, or equivalently concentration of contents or density, of particles in general.
2. Description of the Prior Art
Variations in the morphological characteristics of red blood cells in a patient's blood sample provide valuable information concerning the pathological condition of many specific types of red cell disorders or anemias. Variations in size and color of individual red cells are highly correlated with their volume and hemoglobin concentration. In diagnosing such disorders, the mean cellular hemoglobin concentration (MCHC) and the mean cell volume (MCV) have also been measured to provide valuable insight into the condition of a patient. Such information is usually used in conjunction with the microscopic evaluation of the distribution of sizes, shapes and color of red cells in a stained blood smear by a trained hematologist and with other biochemical tests. For example, in mycrocytic anemias, the size of the red cells and, therefore, also the MCV are significantly reduced (microcytes) but the color and the MCHC are somewhat elevated. In megoblastic anemias, both the size (macrocytes) and the MCHC are somewhat increased.
Recent advances in cytology have produced numerous equipments for automatically measuring the characteristics of red cells, so as to cope with heavy laboratory workloads and with the spiraling increase in medical costs. The better known of such equipments, which are flow cytometers, are the TECHNICON H-6000 system (Technicon Instruments Corporation), the ORTHO ELT-8 system (Ortho Diagnostics) and the Coulter Model "S" system (Coulter Electronics, Dade, Fla.). All of these systems lyse a subsample of blood and measure the optical density of the solution to determine the whole blood hemoglobin concentration (HGB). Also, these systems provide methods for determining other red cell parameters but each based on a different measurement technique. In the TECHNICON H-6000 and the ORTHO ELT-8 systems, individual red cells are passed successively in suspension through a beam of light and the intensity of light scattered within a single angular interval by each such cell is detected and measured as a measure of cell size. The total number of such signals from a fixed volume of unlysed blood also provides the red blood cell count (RBC). The technique used in the TECHNICON H-6000 system for measuring the volume of a red blood cell relates cell volume to the intensity of light scattered by the cell. The intensity of the light scattered by a red blood cell is also dependent upon the refractive index of the cell which is almost entirely influenced by the concentration of hemoglobin in the cell. The hemoglobin and water account for about 99% of the cell contents. Thus, typically, the value of MCV of a sample of red blood cells which is calculated from a measurement of light scattered within a single angular interval depends also on the MCHC of the sample. In the Coulter Model "S" system, an electrical measurement is made whereby each cell is passed in turn through an orifice and the change in electrical resistance across such orifice is a measure of cell size. In the Coulter Model "S" system, a problem with MCHC interference is also present. The cells passing through the orifice are each subjected to significant hydraulic shear, so as to be deformed into an elongated and uniform shape. However, the amount of deformation of the cells is a function of the cell hemoglobin concentration, since it affects the cell viscosity. In a manner similar to that in the TECHNICON H-6000 system, the RBC is determined. Each of these systems accumulates the measurements which are then processed electronically for calculation of MCV which is proportional to the sum of the amplitudes of such measurements on individual cells divided by the number of cells measured. The packed cell volume (HCT) is calculated as the product of RBC and MCV; the MCHC is calculated by dividing HGB by HCT; and the mean cellular hemoglobin content (MCH) is calculated by dividing HGB by RBC. Each of these systems gives the mean measurement of the values of V and HC, that is, MCV and MCHC, respectively, and records the volumes of separate red cells as a distribution curve or histogram. However, such systems are incapable of determining HC on a cell-by-cell basis. Accordingly, an automated measurement of abnormal color variations on a cell-by-cell basis by flow cytometry has not been heretofore available to the diagnostician.
Techniques are known for simultaneously measuring the intensity of the light scattered in the forward direction and the intensity of the light absorbed by individual red blood cells in flow cytometry systems. The former measurement is used to estimate the volume of the cell and the latter measurement is used to estimate the hemoglobin content of the red blood cell. Such a sysem is described in the article "Combined Blood Cell Counting and Classification with Fluorochrome Stains and Flow Instrumentation" by H. M. Shapiro et al, Journal of Histochemistry and Cytochemistry, Vol. 24, No. 1, pp 396-411. An accurate measure of hemoglobin content can be determined by a light absorption measurement only if the index of refraction of the suspending medium matches the index of refraction of the red cells. Then the measurement will be free of an interfering pseudo-absorption scatter signal (see "The Photometric Chemical Analysis of Cells" by A. W. Pollister and L. Ornstein in Analytical Cytology (R. Mellors, ed.) p. 431, McGraw-Hill, New York, 1959). But then the red cells would not scatter light and the volume information would be lost. Therefore, in measurements such as those of Shapiro et al, where the indices of refraction have not been matched, each of the two measurements in fact depends upon both the volume and the index of refraction of the red blood cell being measured. Accordingly, this technique does not achieve an independent measurement of these cell parameters. Also, since the red blood cells being measured in such system are not sphered, the measurements obtained are not accurate.
Also, techniques are known for using image processing and pattern recognition technology to classify red blood cells. One such system is described in U.S. Pat. Nos. 3,851,156 and 4,199,748 and in the article "Image Processing for Automated Erythocyte Classification" by J. W. Bacus, the Journal of Histochemistry and Cytochemistry, Vol. 24, No. 1. pp. 195-201 (1976). In such system, the sample is prepared as a monolayer of dried and flattened cells on a glass microscope slide and the images of individual red cells are analyzed by a microscope image processing and pattern recognition system, each cell being classified by appropriate logic circuits into a distinct subpopulation. Both the distributions of individual cell parameters as well as their means can be determined. However, the volume measurements are computed as being proportional to cell areas because cell thickness cannot be readily measured. However, since cell thickness in such preparations can vary, the above assumption is often in error. Such systems are also slower than flow cytometers, whereby fewer red blood cells per sample can be analyzed per unit time. Therefore, the results are somewhat degraded with respect to flow cytometers.
Further, additional techniques are known for measuring forward scattered light signals to determine the size of particles. For example, in "Particle Sizing by Means of the Forward Scattering Lobe" by J. Raymond Hodkinson, Applied Optics, Vol. 5, 1966, pp. 839-844 (also see P. F. Mullaney and P. N. Dean, Applied Optics, Vol. 8, p. 2361, 1969), the ratio of signals detected at two angles within the forward scattering lobe, or first maximum of the angular distribution of the scattered light, is measured to determine particle size. However, such techniques have been limited to the measurement of only a single parameter, i.e., size or volume, of the particles, and do not apply to the simultaneous measurement of size and index of refraction as do the methods to be described below.